Novel culture systems for EX vivo development

ABSTRACT

The present invention provides methods for the culture of animal pluripotent stem cells and their differentiated progeny cells, tissues, and organs, and nonhuman animal embryos and fetuses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication Ser. No. 60/534,447, filed Jan. 2, 2004 and U.S. ProvisionalApplication Ser. No. 60/539,796, filed Jan. 28, 2004 which areincorporated herein by reference.

FIELD OF THE INVENTION

This invention generally relates to cells, tissue, and organ culturetechnology. More particularly, the invention relates to methods forculturing and differentiating animal pluripotent stem cells andnon-human mammalian embryos and fetuses.

BACKGROUND OF THE INVENTION

Advances in nuclear transfer and embryonic stem cell technology havefacilitated the cloning of non-human animals for diverse applicationsincluding agriculture, xenotransplantation, disease models, recombinantprotein production, and novel means of manufacturing human cells for usein medical therapies, diagnosis, and discovery research. Each of thesepractical applications would benefit from new technologies to improveefficiencies in the production of animals, tissues, and cells. In thecase of animal cloning, the high cost of recipient females to gestatethe cloned fetuses often makes the commercialization of cloned animalsimpractical. In the case of the therapeutic uses of pluripotent stemcells, many pluripotent cells such as human embryonic stem (hES) cells,are problematic to culture using traditional cell culture technology.The cells are dependent on a close association with similarundifferentiated cells and often require being cultured in juxtapositionwith embryonic fibroblast feeder cells in order to maintain them in theundifferentiated state.

In addition, while some cells such as hES cells have a demonstratedpotential to differentiate into any and all of the cell types in thehuman body including complex tissues, and while genes expressed uniquelyin many differentiated cell types are known allowing genetic selectionand purification of populations of any cell type of interest,nevertheless, there is need for new technologies to influence thedifferentiation of pluripotent stem cells such as hES cells, new meansof allowing the cells to differentiate in a three dimensional tissueculture environment, and novel means of purifying the target cells ofinterest, and techniques such as these that can be performed in SPFconditions to minimize the risk of pathogen transmission into humans.

In the field of the cultivation of human cells for human cell therapy,regulatory agencies require production methods wherein the cells aregrown in defined conditions with stringent control over contact of thecells (or anything that may come in contact with the cells) withuncharacterized materials that are a potential source of pathogens. Inthe case of human embryonic stem (hES) cells, it is desirable toidentify a means of cultivating the cells in pathogen-free conditions,differentiating downstream progeny of the cells, scale up the number ofthe cells for batch production, cryopreservation, and geneticmodification.

The original culture of hES cells as reported by Thomson et al (Science.1998 Nov. 6;282(5391):1145-7) was accomplished by culturing the innercell mass of human blastocysts in co-culture with feeder layer ofembryonic murine fibroblasts under culture conditions well known in theart of tissue culture to generate ES cell lines. The murine fibroblastsprovide largely uncharacterized factors that promote the growth of EScells while maintaining them in an undifferentiated state. However, theembryonic murine fibroblasts are also a potential source of pathogensincluding uncharacterized retroviruses. Therefore, novel means ofisolating, culturing, and differentiating hES cells and other cells areof great practical value. While avian CEFs have been shown to supportthe growth of murine ES cells (Yang & Petitte, 1994), and the use ofavian cytokines has been described in in non-human mammalian embryonicstem cell culture, (Poultry Science 73: 965-974), there has been nodescription of the possibility that avian CEFs could be useful inproviding SPF support for the growth of other mammalian ES cells such ashES cells.

In addition, because of the innate capacity of hES cells to organizeinto complex three dimensional tissues including organogenesis, andbecause the growth of tissues in culture systems beyond the size ofapproximately 0.5 mm in thickness is impractical without a means ofsupplying vascular support, there is a need for developing conditionsthat allow for the growth of solid tissues and conditions that providesuitable vascular support for such growing tissue with a dimensions ofgreater than 0.5 mm while maintaining the cells in a specificpathogen-free environment.

The avian egg is a relatively well-characterized structure that hasevolved as a means of providing physiological support to a developingvertebrate embryo, including nutritional support, waste disposal, andgas exchange. The ovum of avian species such as the domestic chicken(Gallus domesticus) is that part of the egg commonly called the “yolk”(FIG. 1). The bulk of the ovum is a colloidal suspension of nutrientswhile a small volume of cytoplasm is concentrated in a regionapproximately 3 mm in diameter called the blastodisc on the animal pole.Following fertilization, the ovum traverses the oviduct acquiringalbuminous material (egg white) and finally the shell membrane and thecalcified egg shell.

In the case of an egg that has become fertilized by sperm subsequent toovulation and prior to encapsulation into the shell, the blastodisc willundergo repeated rounds of karyokinesis and cytokinesis until at aboutthe time the egg is laid, a collection of cells called the blastodermhas formed that is roughly equivalent to the stage of mammalian embryosat the blastocyst stage. Therefore, cultured avian blastodermal cellsare occasionally referred to as avian embryonic stem cells (aES cells)and those from species of domestic chicken are referred to as chickenembryonic stem (cES) cells (U.S. Pat. No. 5,340,740). Following theformation of primitive germ layers of the avian embryo proper,extraembryonic membranes begin to form that will function to support thedeveloping embryo. As shown in FIG. 2, these include the splanchnopleurethat will form the yolk sac, the somatopleure, that will form the amnionand the chorion, and the allantoic membrane, that will eventually fusewith the chorion to form the chorioallantoic membrane. These membranesbecome vascularized and provide the developing embryo with nutrientsfrom the yolk sac and gas exchange across the egg shell.

In contrast to avian species, mammalian development is viviparous andoften occurs in the context of the uterus, where embryonic membranesform analogous to that in the avian egg, but the extraction of nutritionfrom the maternal circulation can occur either through either thechorion, the allantoic membrane, or the yolk sac membrane depending onthe mammalian species. Generally speaking, in most mammals, the yolk sacprovides little if any nutritional support.

The avian egg provides an unusually promising environment for thecultivation of human cells. As described herein, novel means ofculturing and maintaining hES cells, hED cells, and cells differentiatedfrom such cells are described utilizing telolecithal or eutelolecithaleggs or cells derived from embryonated telolecithal or eutelolecithaleggs. In addition, it is possible to utilize telolecithal oreutelolecithal eggs to support the in ovo development on non-humanmammalian embryos and fetuses and to reconstitute embryonic stem cellsand embryo-derived cells from chromatin from mammalian species.

SUMMARY OF THE INVENTION

The present invention provides methods for the culture of animalpluripotent stem cells and their differentiated progeny cells, tissues,and organs, and nonhuman animal embryos and fetuses.

More specifically, this invention provides a novel method of culturingembryos, fetuses, cells, tissues, and organs in ovo in telolecithal oreutelolecithal eggs and for the culture of hES cells, hED cells, andcells differentiated from such cells in co-culture with cells derivedfrom embryonated telolecithal or eutelolecithal eggs for numerouscommercial applications that improves yield, efficiency, cost, and riskin each of the above categories.

In one aspect of the invention, the method comprises: the utilization ofan unfertilized telolecithal or eutelolecithal egg of the avian oregg-laying mammal species as a culture system for the growth anddifferentiation of mammalian stem cells.

In another aspect of the invention, stem cells are implanted within thevitelline membranes of the telolecithal or eutelolecithal oocyte andsubsequently incubated to allow the differentiation of mammalianextraembryonic membranes whereby a mammalian yolk sac splanchnospleuricmembrane surrounds the avian yolk.

In still another aspect of the invention, mammalian embryonic cells canbe injected in ovo in juxtaposition to the vitelline membrane andincubated over time to allow the formation of a plurality of mammalianextraembryonic membranes in the avian egg, including the formation ofmammalian splanchopleure, somatopleure, chorionic membrane (CAM),allantoic membrane, amniotic membrane, or yolk sac membranes. Thegeneration of such extraembryonic membranes has great utility insupporting the differentiation of hES or hED cells for purposes ofresearch or manufacture, or, in the case of non-human mammalian species,in supporting advanced development of embryos and fetuses for researchor production of agricultural animals.

In yet another aspect of the invention, mammalian embryonic cells (suchas hES cells or hED cells, or cells differentiated from such cells) canbe injected in ovo in juxtaposition to the vitelline membrane of anembryonated avian egg to produce differentiated cells vascularized bythe vitelline vascular plexus.

In another aspect of the invention, mammalian pluripotent stem cells(such as hES cells or hED cells, or cells differentiated from suchcells) are injected in juxtaposition with the CAM, or in a region of theegg in which the CAM will eventually invade. The vasculature of the CAMthen supplies vascularization to the growing and differentiating mass ofcells.

In still yet another aspect of the invention, mammalian pluripotent stemcells are injected in the amniotic cavity, albumin, air space, allantoiccavity, extraembryonic coelom, or the yolk sac of the egg and allowed todifferentiate.

In another aspect of the invention, inducers such as factors includinghormones, growth factors, extracellular matrix components, or inducercells are introduced into the avian egg with the stem cells of theabove-mentioned protocols in order to influence the course ofdifferentiation of the injected mammalian pluripotent stem cells.

In a particular aspect of this invention, the inducer cells of thepervious embodiment include avian SPF cells from diverse differentiatedcell lineages including somatic cells obtained from the differentiationof chicken embryonic stem (cES) cells.

In another aspect of the invention, whole and intact nonhuman embryosand fetuses can be cultured in the avian egg with or without a shell orshell membrane (in ovo) through the injection of nonhuman embryos orembryo-derived cells into the egg in juxtaposition to the vitellinemembrane. Whole and intact human embryos could also be developed in ovousing the described invention, however, it is the belief of theinventors that the use of the technology for this purpose is not ethicaland claims for such uses are not sought in the present invention.

In still another aspect of the present invention, intact non-humanmammalian embryos and fetuses can be grown in ovo and used to induce thedifferentiation of injected mammalian pluripotent stem cells includinghES and HEDC cells by injecting such hES, hED, or cells differentiatedfrom such cells into chosen sites of the differentiating non-humananimal embryo or fetus to induce the differentiation of such injectedcells.

In another particular aspect of the invention, nonhuman mammalianembryos and fetuses can be cultivated in ovo by means of the transfer ofchromatin into the blastodisc of an unfertilized avian egg, where theavian oocyte is activated and induced to undergo rounds of karyokinesisand cytokinesis and subsequent development. Human chromatin can also beintroduced into the blastodisc of the avian egg for the purpose ofreconstituting intact embryonic cells from reprogrammed chromatin, butthe development of intact human embryos post gastrulation and fetuses bythis means is considered unethical and claims relating to humanpost-gastrulation embryos or fetuses cultured in ovo are not sought inthis application.

In another aspect of the invention, embryonic cells from SPF speciesincluding SPF embryonic chicken cells are used as feeder cells for thein vitro cultivation of mammalian ES cells including hES and hEDC cellsin vitro or in ovo.

In another aspect of the invention, somatic cells from SPF speciesincluding SPF embryonic chicken cells are used as cells to induce thedifferentiation of hES or hED cells or cells differentiated from suchcells. The SPF inducer cells may be viable or mitotically inactivated byradiation or chemical treatment, and may be co-cultured with the humanstem cells in a variety of culture conditions including in vitro and inovo co-culture.

Other features and advantages of the invention will be apparent from thefollowing description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing illustrating the transfer of stem cells injuxtaposition to the vitelline membrane of the unfertilized or earlyembryonated avian egg. In the example shown, the cells are injected withan inducer to influence the course of differentiation of the stem cells.

FIG. 2 is a drawing of an artificial culture vessel for maintainingmammalian pluripotent stem cells and derivative cells in the presence ofcomponents of a telolecithal or eutelolecithal egg.

FIG. 3 is a drawing of the various anatomical structures of thefertilized chicken egg, showing the location of the chorioallantoicmembrane (CAM) and the placement of mammalian pluripotent stem cells andinducer in juxtaposition to the CAM membrane.

FIG. 4 is a drawing showing the result of placement of mammalian EScells or embryo-derived cells within the vitelline membrane of anembryonated egg such that the growing teratoma is vascularized by thechick's vitelline vascular plexus.

FIG. 5 shows an hematoxylin-and-eosin stained tissue section from ahuman teratoma formed by the placement of human ES cells within thevitelline membrane of an embryonated egg.

FIG. 6 shows the use of SPF chick embryo fibroblasts to stably maintainhES cell lines in an undifferentiated state. Morphology (A-C) andmarkers (D-I) of undifferentiated hES cells grown on CEF: A—colonies ofhES cell line H9 on CEF. B, C—colonies of the hES cell line H1 grown onCEF (B) vs. on MEF (C); D-I, hES cell line H7 cultured on CEF (4passages): D, Oct-4; E, SSEA-3; F, SSEA-4; G, alkaline phosphatase; H,TRA-1-60; I, TRA-1-81. Original magnification: A, x38; B-I, x200

DETAILED DESCRIPTION OF THE INVENTION

Table of Abbreviations

-   [Ca⁺²]i—Intracellular calcium concentration-   CAM—Chorioallantoic membrane-   CEF—Chick Embryo Fibroblast-   cES Cell—Chicken embryonic stem cell-   ES Cell—Embryonic stem cells derived from a morula or    blastocyst-staged mammalian embryo produced by the fusion of a sperm    and egg cell, nuclear transfer, parthenogenesis, or the    reprogramming of chromatin and subsequent incorporation of the    reprogrammed chromatin into a plasma membrane to produce a cell.-   HEDC—Human Embryo-Derived Cells-   hES Cell—Human embryonic stem cells-   ICM—Inner Cell Mass of the blastocyst embryo.-   ICSI—Intracytoplasmic sperm injection-   MII—Metaphase II-   NT—Nuclear Transfer-   SPF—Specific Pathogen-Free

The present invention provides methods for the culture of mammalian stemcells, differentiated progeny cells, tissues and organs, and non-humanmammalian embryos and fetuses in a telolecithal or eutelolecithal eggsuch as that of avian or egg laying mammalian species (in ovo). The term“in ovo” refers to residence within a shelled telolecithal oreutelolecithal egg, or in the presence of the components of such an eggor eggs cultured in a container other than an egg shell, such containerbeing composed of polymers, glass, or metal. The telolecithal oreutelolecithal eggs useful in the present invention may be from thecommon domestic chicken (Gallus gallus domesticus) or from any otheravian species including but not limited to the turkey (Meleagris), quail(Coturnix), and duck (Anas) or an egg-laying mammals such as those ofthe Order Monotremata. The avian eggs useful in this invention for theproduction of therapeutic products include specific pathogen-free (SPF)eggs. The term “specific pathogen-free” refers to eggs that have beenobtained from animals reared in conditions to insure that the animalsand their eggs are free of known pathogens including avian pathogenicviruses.

The term “suicide gene” refers to genes that may be introduced into themammalian stem cells or into the avian inducer cells or into the aviansystem providing vascular support, such that upon stimulation, the cellsthat carry the suicide gene can be induced to die. Such suicide genesare well known in the art and include the use of herpes simplex virusthymidine kinase that in the presence of gancyclovir can cause the deathof the cell carrying the gene. The term “mitotically inactivated” refersto cells that have been rendered incapable of subsequent cell divisionby the exposure of such cells to agents that damage the DNA of suchcells such that the cells undergo DNA damage checkpoint arrest orapoptosis. Such mitotic inactivation can be achieved by techniques wellknown in the art such as the use of exogenous radiation, or chemicalagents including mitomycin C.

The term “teratoma” refers to a benign mass of cells differentiatingfrom pluripotent stem cells that organize into complex tissues in threedimensions, though lacking the normal and intact form of an animal andincapable of independent life. By way of example, teratomas have beenreported to occur following the injection of hES cells into the skeletalmuscle or peritoneum of immunocompromised mice where such teratomascontain intenstine, skin, teeth, renal tissue, neuronal tissue, bone,cartilage, and so on.

The term “chorioallantoic membrane” or “CAM” refers to the outermostextraembryonic membrane that eventually lines the noncellular eggshellmembrane. The CAM is formed by the fusion of the splanchnic mesoderm ofthe allantois and the somatic mesoderm of the chorion. The fused doubletof allanois and chorion will cover the entire inner surface of the eggshell by day 12.

The term “pluripotent stem cells” refers to animal cells capable ofdifferentiating into more than one differentiated cell type. Such cellsinclude hES cells, hEDCs, and adult-derived cells including mesenchymalstem cells, neuronal stem cells, and bone marrow-derived stem cells.Pluripotent stem cells may be genetically modified or not geneticallymodified. Genetically modified cells may include markers such asfluorescent proteins to facilitate their identification within the egg.

The term “embryonic stem cells” (ES cells) refers to cells derived fromthe inner cell mass of blastocysts or morulae that have been seriallypassaged as cell lines. The ES cells may be derived from fertilizationof an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, orby means to generate hES cells with homozygosity in the MHC region. Theterm “human embryonic stem cells” (hES cells) refers to cells derivedfrom the inner cell mass of human blastocysts or morulae that have beenserially passaged as cell lines. The hES cells may be derived fromfertilization of an egg cell with sperm or DNA, nuclear transfer,parthenogenesis, or by means to generate hES cells with homozygosity inthe HLA region.

The term “human embryo-derived cells” (HEDC) refer to morula-derivedcells, blastocyst-derived cells including those of the inner cell mass,embryonic shield, or epiblast, or other totipotent or pluripotent stemcells of the early embryo, including primitive endoderm, ectoderm, andmesoderm and their derivatives, but excluding hES cells that have beenpassaged as cell lines. The hEDC cells may be derived from fertilizationof an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, orby means to generate hES cells with homozygosity in the HLA region.

In one embodiment of the invention, mammalian pluripotent stem cellswith or without inducer molecules or cells are injected within and injuxtaposition to the vitelline membranes of the unembryonated egg (FIG.1). One mammalian pluripotent cell, or a plurality of cells, forexample, a colony of cultured mammalian pluripotent stem cells such asES cells, in particular hES or HEDC cells, can be injected by techniqueswell known in the art, such as incubating an egg at 37-39° C. in 60%humidity, the shell cleaned with 70% ethanol, and using a sterilesyringe, approximately 2.5 mL of albumin will be removed. This allows asmall, typically 1.5 cm² window in the shell to be made and cells to beinjected with a glass pipette, and subsequent covering the windowedportion of the shell with a sealant such as common kitchen wrap andsubsequent culture at 37° C. with or without supplemental calcium andascorbate to approximate the physiological levels of the correspondingmammalian species in a standard tissue culture incubator. The egg may beinjected at one site, or multiple sites, including at or near theblastodisc, depending on the nature of the cells and the type of productdesired.

In addition, the cells with or without inducer may be injected withinthe vitelline membrane but external to the developing embryo of anembryonated egg such that the differentiated cells are vascularized bythe vitelline vascular plexus. The differentiated cells can then beremoved from the egg and purified from the yolk sac prior to hatching.Alternatively, the chicken can be allowed to develop to hatching, inwhich case the yolk sac membrane is absorbed within the body cavity ofthe chick and the mammalian teratoma continues to develop within thebody of the hatched chick and the differentiated mammalian cells can beremoved post hatch. Some of the advantages of obtaining the cells posthatch are that it allows more time for greater growth and development ofthe teratoma and it provides early exposure of the chick to themammalian pluripotent stem cells which tolerizes the immune system andlessens chances of rejection. As in the case of injection of cells intounembryonated eggs, the injection of the cells into embryonated eggs isby techniques well known in the art for the injection of cells, such asthe injection of avian blastodermal cells into the blastoderm of afertilized egg to generate chimeras. The egg is cultured at 37° C. or inthe proximity to the normal temperature for human cells (i.e. 35-39° C.)at about 60% humidity, the shell cleaned with 70% ethanol, and using asterile syringe, approximately 2.5 mL of albumin will be removed. Thisallows a small, typically 1.5 cm² window to be made in the shell for theintroduction of cells with or without supplemental calcium and ascorbateto approximate the physiological levels of the corresponding mammalianspecies.

In another embodiment of the invention, the components of the egg willbe transferred to a container such as that shown in FIG. 2 to replacethe function of the egg shell and to facilitate the manipulation of theculture system. Such container may contain a transparent component toallow the viewing of the developing tissue, ports for the removal,replacement, or addition of egg components such as egg albumin or aculture medium or matrix substrate substituting for albumin, egg yolk,mammalian pluripotent stem cells including hES or HEDC, or inducermolecules or cells, the cannulation of blood vessels within thedifferentiating tissue for external circulatory or respiratory support,or a system such as a semipermeable membrane to facilitate the diffusionof gases and small molecules into and out of the culture system. The useof an artificial container also allows for the introduction of eggcomponents from multiple eggs for culture of cells of animals of longgestational age and where larger tissues or larger extraembryonicmembranes are desired, with or without supplemental calcium andascorbate to approximate the physiological levels of the correspondingmammalian species.

In another embodiment of the invention, mammalian pluripotent stem cellsincluding hES and hEDC cells are injected in the proximity of the shellmembrane to form a teratoma that will subsequently become vascularizedby the growing CAM membrane (FIG. 3). Typically, in the case of thechicken egg, the egg will be incubated at approximately 37° C. and 60%humidity, the shell cleaned with 70% ethanol, and using a sterilesyringe, approximately 2.5 mL of albumin will be removed. This allows asmall, typically 1.5 cm² window to be cut in the shell and the shellmembrane allowing the mammalian pluripotent cells to be injected withinthe albumin and in juxtaposition to the shell membrane. The mammalianpluripotent cells may be injected between day 1 and day 17. The teratomamay subsequently be removed and cultured in organ culture with theattached vasculature used to perfuse the growing tissue with blood ortissue culture media. Any residual avian cells may be removed byactivation of the avian suicide genes.

In another embodiment of the invention, mammalian pluripotent stem cellsincluding hES and HEDC are injected by the above techniques in theamniotic cavity, albumin, air space, allantoic cavity, extraembryoniccoelom, or the yolk sac of the egg and allowed to differentiate overtime in the incubated egg.

In another embodiment of the invention, the inducer includes cells thatare derived from cells of a heterologous species, such as chickensomatic cells inducing the differentiation of hES cells. Such cells canbe cells that normally occur in juxtaposition to the cell of interestand include stromal cells and endothelial cells from the organ orparenchyma of interest. The somatic inducer cells can be obtained from avariety genotypes including SPF eggs to reduce the risk of pathogentransmission. Such eggs are commercially available (Charles RiverLaboratories) and are free of such pathogens as Avian AdenovirousesI-III, Avian Encephalomyelitis, Avian Influenza (Type A), AvianNephritis Virus, Avian Paramyxovirus Type 2, Avian Reovirus, AvianRhinotracheitis Virus, Avian Rotavirus, Avian Tuberculosis, ChickenAnemia Virus, Endogenous GS Antigen, Fowl Pox, Hemophilusparagallinarum, Infectious Bronchitis (Ark, Conn, JMK, and Mass),Infectious Bursal Disease, Infectious Laryngotracheitis, LymphoidLeukosis A,B, Lymphoid Leukosis Viiruses, Marek's Disease (Serotypes1,2,3), Mycoplasma gallisepticum, Mycoplasma synoviae, NewcastleDisease, Reticuloendotheliosis Virus, Salmonella pullorum-gallinarum,and other Salmonella species.

In another embodiment of the invention, the inducer cells are derivedfrom ES cells of a heterologous species. By way of non-limiting example,the inducer cells may be cES cells differentiated into somatic cellsthat function in inducing the specific differentiation of hES cells. ThecES cells can be obtained from a variety genotypes including SPF eggs toreduce the risk of pathogen transmission. In addition, since the cEScells can be cultured indefinitely in an undifferentiated state, theycan be genetically modified using techniques well known in the art forimproved performance as inducer cells. Such genetic modificationsinclude the introduction of suicide genes that allow the destruction ofthe inducer cells prior to use, modified to express cell surfaceantigens that facilitate the removal of the inducer cells by affinitymethods well known in the art, or the inducer ES cells may be modifiedby gene trap vectors in order to obtain ES cell clones that expressmarkers such as fluorescent proteins that facilitate the purificationand identification of particular differentiated cell types as inducercell lines.

In another embodiment of the invention, the inducer is one of a numberof extracellular signaling molecules including growth factors,cytokines, extracellular matrix components, nucleic acids encoding theforegoing, steroids, and morphogens or neutralizing antibodies to suchfactors. Such inducers include but are not limited to: cytokines such asinterleukin-alpha A, interferon-alpha A/D, interferon-beta,interferon-gamma, interferon-gamma-inducible protein-10,interleukin-1-17, keratinocyte growth factor, leptin, leukemiainhibitory factor, macrophage colony-stimulating factor, and macrophageinflammatory protein-1 alpha, 1-beta, 2, 3 alpha, 3 beta, and monocytechemotactic protein 1-3.

Differentiation agents according to the invention also include growthfactors such as 6kine, activin A, amphiregulin, angiogenin,β-endothelial cell growth factor, β-cellulin, brain-derived neurotrophicfactor, C10, cardiotrophin-1, ciliary neurotrophic factor,cytokine-induced neutrophil chemoattractant-1, eotaxin, epidermal growthfactor, epithelial neutrophil activating peptide-78, erythropioetin,estrogen receptor-alpha, estrogen receptor-beta, fibroblast growthfactor (acidic and basic), heparin, FLT-3/FLK-2 ligand, glial cellline-derived neurotrophic factor, Gly-His-Lys, granulocyte colonystimulating factor, granulocytomacrophage colony stimulating factor,GRO-α/MGSA, GRO-β, GRO-gamma, HCC-1, heparin-binding epidermal growthfactor, hepatocyte growth factor, heregulin-alpha, insulin, insulingrowth factor binding protein-1, insulin-like growth factor bindingprotein-1, insulin-like growth factor, insulin-like growth factor II,nerve growth factor, neurotophin-3,4, oncostatin M, placenta growthfactor, pleiotrophin, rantes, stem cell factor, stromal cell-derivedfactor 1B, thrombopoietin, transforming growth factor- (alpha,beta1,2,3,4,5), tumor necrosis factor (alpha and beta), vascularendothelial growth factors, and bone morphogenic proteins.

Differentiation agents according to the invention also include hormonesand hormone antagonists such as 17B-estradiol, adrenocorticotropichormone, adrenomedullin, alpha-melanocyte stimulating hormone, chorionicgonadotropin, corticosteroid-binding globulin, corticosterone,dexamethasone, estriol, follicle stimulating hormone, gastrin 1,glucagons, gonadotropin, L-3,3′,5′-triiodothyronine, leutinizinghormone, L-thyroxine, melatonin, MZ-4, oxytocin, parathyroid hormone,PEC-60, pituitary growth hormone, progesterone, prolactin, secretin, sexhormone binding globulin, thyroid stimulating hormone, thyrotropinreleasing factor, thyroxin-binding globulin, and vasopressin.

In addition, differentiation agents according to the invention includeextracellular matrix components such as fibronectin, proteolyticfragments of fibronectin, laminin, tenascin, thrombospondin, andproteoglycans such as aggrecan, heparan sulphate proteoglycan,chontroitin sulphate proteoglycan, and syndecan. Such extracellularmatrix components may be injected at or near the site of the injectedpluripotent stem cells in a soluble form or attached to an immobilizedmatrix such as a tissue membrane or a membrane made of a syntheticpolymer.

Differentiation agents according to the invention also includeantibodies to the previously-mentioned cytokines, growth factors,hormones, and extracellular matrix components, and their receptors.

The present invention also provides for a means of developing humanextra-embryonic membranes that can function to support the near normaldifferentiation of cells from hES cells or hED cells in ovo. Theinjection of such human pluripotent stem cells such as hES cells at ornear the vitelline membrane of either an embryonated or unembryonatedegg by injection and subsequent incubation techniques well known in theart and described in application above, results in the differentiationof some of the injected cells into extra-embryonic membranes such ashuman amnion, chorion, and yolk sac, that in turn provide laboratorymodels of cell differentiation, and the derivation of yolk sachematopoietic precursor cells, and extra-embryonic membranes useful insupporting the growth and differentiation of such stem cells.

The present invention also provides for a means of developing mammalianextra-embryonic membranes that can function to support the near normaldevelopment of non-human fetuses. In particular, such species as thedomestic pig display extra-embryonic membrane formation closelyresembling that of avian species and such animals can be gestated withinthe avian egg or within an artificial device such as that shown in FIG.2. The inner cell mass or embryonic disc or embryonic stem cells of suchnon-human mammalian preimplantation embryo or peri-implantation embryocan be grafted into or near the blastodisc of an unfertilized avian egg,or the blastoderm of a fertilized avian egg can be removed orinactivated and replaced by the intact ICM or embryonic disc of anon-human mammalian embryo with or without supplemental calcium andascorbate to approximate the physiological levels of the correspondingmammalian species

The present invention also provides a means of influencing thedifferentiated state of cultured hES cells, hED cells, and cellsdifferentiated from such cells by co-culturing such cells with SPF aviandifferentiated cells. SPF avian chick embryo fibroblasts, including butnot limited to chick embryo fibroblasts from SPF embryonated eggs atnine days of culture may be isolated by techniques well known in the artsuch as by removing such nine day-old chick embryos, disaggregating thetissues, and plating the cells in standard fibroblast growth conditionssuch as MEM medium supplemented with 10% FBS or defined pathogen-freemedium. hES cells may then be serially passaged onmitotically-inactivated SPF chick embryo fibroblasts instead of usingfeeder cells such as murine embryo fibroblasts with an uncharacterizedpathogen status. The co-culture of hES cells with SPF chick embryofibroblasts has a clear utility in facilitating the scale up of hEScells in pathogen-free culture conditions. The use of other specific SPFchick cells may similarly be used where such cells are known to causethe induction of differentiation in order to influence thedifferentiation of hES, hED cells, or other downstream pluripotent humancells. Examples of cell types that function as inducers ofdifferentiation are well known in the art and include mesodermal cellssuch as the stromal cells from the aorta-gonadal-mesonephros regionwhich induce definitive hematopoiesis in pluripotent stem cells,ectodermal cells such as the optic vesicle cells, or mesenchymal cellsfrom the optic vesicle that induce the differentiation of ectodermalcells into lens cells, and endodermal cells such as the induction ofpancreatic islet cells, including pancreatic beta cells from primitiveendodermal epithelium by pancreatic mesenchymal cells. Induction canalso occur by epithelio-stromal interactions and by the use of onegerm-layer to induce cells of another germ-layer, such as the use ofdermal mesoderm cells to induce epidermal differentiation such as hairdifferentiation, mesodermally-derived cells that induce gut andultimately pancreatic islet cell differentiation, the mesodermal cellsof the ureteric bud that induce kidney differentiation, the mesodermalinduction of epithelium to produce pharyngeal thymus and thyroiddifferentiation, liver mesenchymal cells that induce primitiveepithelium to differentiate into hepatic cords and liver parenchyma, gutmesenchymal cells that induce primitive epithelial cells todifferentiate into gut, tracheal mesenchymal cells that inducerespiratory differentiation such as respiratory epithelium, Such inducercells can be removed from the corresponding region of an SPF chickembryo by standard dissection, or isolated from SPF chick ES cell linesutilizing genetic markers for that lineage of cells, such as exogenousmarkers with exogenous promoters or using the endogenous promoter andgene trap technology.

The present invention also provides a means of reconstituting mammaliancells from chromatin, by removing or inactivating the avian DNA from theblastodisc of an avian embryo using techniques well known in the art,and replacing said genome with the haploid or preferably the diploidgenome of a mammalian cell. The mammalian cell genome may be by way ofexample, human somatic cell-derived chromatin that has been reprogrammedand condensed by exposure in vitro to extracts or purified componentsfrom metaphase II oocytes as is known in the art. Subsequent or at aboutthe time of the transfer of chromatin, the oocyte is activated such thatthere is an elevation of intracellular calcium. Current strategies forthe activation of the oocyte in the absence of sperm, commonly known asparthenogenetic activation are well known in the art, and includechemical activation to elevate intracellular calcium concentrationfollowed by the down-regulation of maturation-promoting factor (MPF),the injection of sperm extracts or purified sperm factor, or incubationin strontium chloride. In addition, this invention provides a novelmethod of activating the oocyte of a telolecithal or eutelolecithal eggin conjunction with the transfer of chromatin from a mammalian species,said method being the injection and subsequent removal of a sperm,multiple sperm, or sperm heads, and their subsequent removal. As aresult of chromatin transfer and activation, rounds of karyokinesis andcytokinesis that follow result in cells similar in nature to hES or HEDCcells on the in juxtaposition to the vitelline membrane as previouslydescribed.

Applications

It is envisioned that the disclosed methods for the culture of animaltissues are generally useful in mammalian subjects, including human andnon-human subjects, and particularly in the culture of non-human embryosand fetuses and for the culture and differentiation of mammalianpluripotent stem cells, in particular, hES cells and HEDC.

Following a review of the present disclosure, one skilled in the art ofstem cell culture and the manipulation of telolecithal eggs such asavian eggs, can readily implement the invention in the culture ofnon-human mammalian embryos and fetuses, and in the culture of mammalianstem cells including human stem cells. As described further hereinbelow, the methods of the present invention can be used for culturingnon-human embryos such as pigs to advanced stages of development, andfor the manufacture of animal cells, such as human cells useful in drugdiscovery, basic research, and in cell therapy.

A. Development of Mammalian Cells and Tissues in the Avian Egg

In one embodiment of the invention, hES cells and other mammalian EScells are cultured and differentiated within the avian egg. The term“avian egg” refers to the fertilized or unfertilized egg of an avianspecies including but not limited to eggs of the domestic chicken(Gallus domesticus), the turkey, duck, ostrich, and quail. However,complex tissues can be produced using this invention, similar to theproduction of teratomas which are disorganized aggregations of humantissue that form after the injection of human embryonic stem cells intoimmunocompromised mice.

The resultant differentiated progenitor cells or fully differentiatedcells of the present invention, preferably human differentiated cells,have numerous therapeutic and diagnostic, and basic researchapplications. Most specifically, such differentiated cells may be usedfor cell transplantation therapies. Human differentiated cells haveapplication in the treatment of numerous disease conditions.

The subject differentiated cells may be used to obtain any desireddifferentiated cell type. Therapeutic useages of such differentiatedcells are unparalleled. For example, human hematopoietic stem cells andhemangioblasts may be used to treat many diseases that compromise theimmune system, such as AIDS, cancer therapy, or age-related immunedysfunction. Hematopoietic stem cells can be obtained, e.g., by fusingadult somatic cells of a cancer or AIDS patient, e.g., fibroblasts orblood cells with an enucleated oocyte, obtaining inner cell mass cells,and culturing such cells in ovo under conditions which favordifferentiation until hematopoietic stem cells or hemangioblasts areobtained. By way of a non-limiting example, hES or HEDC cells orprimitive mesodermal cells derived from such cells can be injected inovo using one of the techniques described herein in conjunction withstromal fibroblasts from the aorta-gonadal-mesonephros region of anon-human mammalian embryo or fetus or avian species to induce thedifferentiation of the cells into hemangioblasts and hematopoietic stemcells. Such cells may then be used with or without genetic modificationfor the treatment of diseases including AIDS, cancer, and immunedysfunction. The cells can also be used in veterinary practice to treatcanine or feline disease using cell therapy.

Alternatively, adult somatic cells from a patient with a neurologicaldisorder may be fused with an enucleated oocyte, human inner cell masscells obtained therefrom, and such cells cultured in ovo underdifferentiation conditions to produce neural cell lines and neuralprogenitor cells lines. Specific diseases treatable by transplantationof such human neural cells include, by way of example, Parkinson'sdisease, Alzheimer's disease, ALS, palsy, and spinal cord injury amongothers. In the specific case of Parkinson's disease, it has beendemonstrated that transplanted fetal brain neural cells make the propersynapses with surrounding cells and produce dopamine. This can result inlong-term reversal of Parkinson's disease symptoms and diseaseprogression.

The great advantage of the subject invention is that it provides anessentially limitless supply of isogenic or homozygous MHC cellssuitable for transplantation. Therefore, it will obviate the significantproblem associated with current transplantation methods, i.e., rejectionof the transplanted tissue which may occur because of host-vs-graft orgraft-vs-host rejection. Conventionally, rejection is prevented orreduced by the administration or anti-rejection drugs such ascyclosponine. However, such drugs have significant adverse side-effects,e.g., immunosuppression, carcinogenic properties, as well as beingcostly. The present invention will eliminate, or in the case ofhomozygous MHC cells, greatly reduce the need for anti-rejection drugs.

In addition, the present invention provides a means of directlydifferentiating cells in the context of a SPF culture system capable ofgenerating complex tissues. It also allows for the introduction ofinducer molecules and cells from similar or identical SPF species todirect the differentiation of the cells without the complication ofpathogen transmission from murine or other retroviruses or other unknownagents.

In addition, the present invention provides methods to culture mammalianteratomas near the CAM of an embryonated telolecithal or eutelolecithalegg such that the teratoma is provided vascular support from thedeveloping chick. Such a teratoma can be later removed from the egg andcannulated to provide a growing and vascularized three-dimensionaltissue. Since many complex tissues are limited by the rate of diffusionof gases such as oxygen and carbon dioxide and the exchange of nutrientsand waste products, the ability to assemble three dimensional aggregatesof cells derived from such cells as hES and HEDC cells with vasculatureis an important and novel advance facilitating the production of suchtissues as renal tissue, heart tissue, liver tissue, pancreatic tissue,lung, as well as many other tissue types with dimensions in excess of0.5 mm in diameter.

B. The Transfer and Development of Non-Human Mammalian Embryos In Ovo

In another embodiment of the invention, whole and intact non-humanmammalian embryos and fetuses are gestated in ovo. This system wouldhave great utility in producing cloned offspring where the relativeinefficiencies and high cost of recipient animals leads to a high endcost of product. Animals such as domestic pigs whose extraembryonicmembranes closely resemble that of the avian embryo and whose placentadoes not form a syncitia with the maternal uterus are especially suitedfor development in ovo. In addition to providing a means of gestatingdomestic animals, genetically modified non-human animals developed inovo provide a sterile and SPF system for producing cells and tissues forxenotransplantation. In addition, the non-human animal developing in ovocan be used as an intact animal to induce the differentiation ofmammalian pluripotent stem cells including hES and HEDC cells. By way ofnon-limiting example, hES or HEDC cells or primitive mesodermal cellsderived from such cells can be injected into theaorta-gonadal-mesonephros region of a non-human mammalian embryo orfetus to induce the differentiation of the cells into hemangioblasts andhematopoietic stem cells.

C. The Transfer of Reprogrammed Bovine Chromatin Into the Blastodisc InOvo

The high value placed on mature human oocytes will lead to improvedtechnologies to remodel the chromatin of human cells in oocyte extracts,or eventually to reprogram human DNA using defined molecular components.Such technology is currently known in the art where the extract isobtained from metaphase II oocytes. The reprogrammed chromatin resultingfrom such reprogramming can be injected into the blastodisc of anunfertilized telolecithal or eutelolecithal egg with resulting rounds ofkaryokinesis and cytokinesis resulting in reconstituted and reprogrammedcells within the vitelline membrane. Such cellular reconstitution,especially where such cells can be subsequently grown and differentiatedin ovo as described in the present invention, provides an efficient andcost-effective means of producing differentiated cells of many kindsunder SPF conditions and would therefore have great utility and value inproducing human and non-human animal cells for basic research, drugdiscovery, and cell therapy.

D. The Co-Culture of SPF Avian Cells and Human Pluripotent Stem Cells InVitro

The present difficulties of differentiating human pluripotent stemcells, such as hES cells into desired differentiated cell types such asdefinitive hemangioblasts, pancreatic islet cells, heart muscleprecursor cells, neural progenitor cells, renal cells, liver cells, lungcells, cartilage cells, or dermal cells demonstrates the need for newtechnologies to direct the differentiation of such pluripotent cells andto grow the cells in a defined pathogen-free culture system. In additionto providing new pathogen-free differentiation conditions, the presentinvention provides a novel mean of expanding hES cells in vitro withfeeder cells that, unlike murine embryo fibroblasts, are known to bepathogen free, thereby allowing the hES cells to be cultured inconditions that assure their being free of exogenous pathogens andtherefore minimizing the risk of transmitting pathogens to patients inneed of such cell therapy.

EXAMPLES Example 1

Human Embryo-Derived Cells Differentiated in Juxtaposition to anEmbryonated Telolecithal Egg

Approximately 10×10⁶ human ES cells were trypsinized from culture, thetrypsin was neutralized with 10% FCS in DMEM and the cells pelleted andresuspended in DMEM. Approximately 1×10⁶ human ES cells were injectedwithin the vitelline membrane of an embryonated SPF egg (Charles River)at two days of incubation at 0.5 cm from the avian embryo. At day 15,the mass of cells were identified beneath the yolk sac membrane, fixed,and Hematoxylin-and-eosin stained. In this example the cells were fixedwith formaldehyde, however there are many fixative agents known to thoseskilled in the art which could be used. As shown in FIG. 5, dense sheetsof cells ranging from vacuolated mesenchymal to round cells werevisible, consistent with a predisposition to teratoma formation. Yolksac associated epithelial cells were also observed.

Example 2

Human Embryonic Stem Cell Lines Maintained in the Undifferentiated StateUsing SPF-Chick Embryonic Feeder Cells

Preparation of CEF:

CEF were isolated from 7-8 day old chicken embryos with the heads lefton, using the previously described techniques for isolation of mouseembryonic fibroblasts. Briefly, the embryos were eviscerated, the headsleft on, digested with trypsin and plated onto gelatin coated plates inDMEM, supplemented with 10% FBS, glutamine and penicillin-streptomycin.The cells were frozen at passage one and used at passage 2 after mitoticinactivation with mitomycin C.

The hES cell lines, H9, H7 (both NIH-approved) and ACT-4 wereconsecutively cultured on CEF for 3-6 passages without significantchanges in undifferentiated morphology or growth rate. Passages used forthe experiment: H-9 & H-7: H-9 started passage 38 through passage 40, H7started 29 and through passage 35; and ACT 4 derived here from passage9-11 and 15-19.

Expression of the markers of pluripotency (Oct-4, alkaline phosphatase,SSEA3, SSEA-4, TRA-1-60, TRA-1-81) remained high in hES cells (line H7)after culturing on CEF for 4 consecutive passages. FIG. 6 shows theundifferentiated hES grown on the CEF.

Example 3

Non-Human Embryonic Development Within a SPF Avian Egg and the Use ofthe Porcine Embryo to Direct the Differentiation of Human PluripotentCells

A cloned or normal porcine blastocyst with or without a transgenicsuicide gene is held with an aspiration pipette under low magnificationand the trophectoderm is torn opposite the inner cell mass to yieldnear-planar aggregation of cells. The torn blastocyst is injected with a200 micron pipette into an unfertilized but fresh SPF windowed avian eggat or near the blastodisc. The resulting reconstructed egg is thenresealed with kitchen wrap as is well known in the art and cultured at37° C. on a racking platform. At the point when cell differentiation ofa desired type is occurring in the porcine embryo, hES or hED cells areinjected into the porcine embryo. In the case of hematopoieticdifferentation, the human pluripotent stem cells are injected into theaortic-gonadal-mesonephros region of the porcine embryo to inducedifferentiation into hematopoietic differentiation such ashemangioblasts.

Example 4

The Use of SPF Avian Mesodermal Cells of the Aorta-Gonadal-MesonephrosRegion to Direct the Differentiation of Human Pluripotent Cells intoHemangioblasts

hES or hED cells are co-cultured with mesenchymal cells dissected fromthe aortic-gonadal-mesonephros region of SPF avian embryos to inducedifferentiation into hematopoietic differentiation such ashemangioblasts. The co-culture is incubated in pathogen-free tissueculture until primitive hemangioblasts are produced which aresubsequently purified by the use of antigens such as CD4, AC133, c-kit,or other antigens well known in the art.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The above specification, examples, and data provide a completedescription of the manufacture and use of the invention. Unlessotherwise specified, all patent and non-patent references cited arehereby incorporated by reference.

1. A method for culturing mammalian pluripotent stem cells, comprisingculturing said cells in an unfertilized telolecithal or eutelolecithalegg.
 2. The method of claim 1, wherein the mammalian pluripotent stemcells are human.
 3. The method of claim 1, wherein the mammalianpluripotent stem cells are canine or feline.
 4. A method for culturingmammalian pluripotent stem cells, comprising culturing said cells in anfertilized telolecithal or eutelolecithal egg external to the developingnon-human animal until the time of the involution of the yolk sac. 5.The method of claim 1, wherein the mammalian pluripotent stem cells arehuman.
 6. The method of claim 1, wherein the mammalian pluripotent stemcells are canine or feline.
 7. A method of culturing a nonhumanmammalian embryo or fetus, comprising culturing said nonhuman mammalianembryo or fetus in an unfertilized telolecithal or eutelolecithal egg.8. The method of claim 4, wherein the mammalian species is porcine.
 9. Amethod of reconstituting mammalian cells from chromatin, comprisinginjecting said chromatin into the oocyte of a telolecithal oreutelolecithal egg, activating the egg, and allowing karyokinesis andcytokinesis to occur.
 10. The method of claim 6, wherein the mammalianchromatin is human.
 11. The method of claim 6, wherein the oocyte isactivated by the introduction and removal of a sperm or sperm head. 12.A method of culturing mammalian pluripotent stem cells comprisingculturing said cells in juxtaposition to the vitelline membrane of anembryonated telolecithal or eutelolecithal egg.
 13. A method ofaffecting the growth and differentiation of human embryo-derived cellscomprising the co-culture of said human embryo-derived cells with cellsof a specific pathogen-free species.
 14. The method of claim 13, whereinthe specific pathogen-free species is avian.
 15. The method of claim 14,wherein the human embryo-derived cells are maintained as embryonic stemcell lines by co-culture with chick embryonic fibroblasts feeder cells.16. The method of claim 14, wherein human embryo-derived cells aredifferentiated under the inductive effect of avian cells.
 17. The methodof claim 16, wherein the avian inducer cells are specific pathogen-free.18. The method of claim 16, wherein the avian inducer cells aredifferentiated cells.
 19. The method of claim 18, wherein the avianinducer cells are mesodermal cells of the aorta-gonadal-mesonephrosregion and are used to induce hematopoiesis.
 20. The method of claim 16,wherein said avian inducer cells are differentiated from avian embryonicstem cells.
 21. The method of claim 20, wherein said avian inducer cellsderived from avian embryonic stem cells are identified using gene trapmarkers.